In particular, Marek's disease has been a problem of the poultry industry from the beginning of intensive production of poultry meat. It is a herpes viral disease that is causing a large variety of clinical signs starting from immunosuppression, neurological disorders, anemia and unspecified apathies and ending with severe lymphatic cancers at later stages of infection. In the beginning of the history of Marek's disease, there were no treatments and no preventive measures. Then an apathogenic-related (Serotype 3) virus was isolated from turkeys (HVT) and was initially used for vaccination.
However, some time after introduction of vaccination with HVT, Marek's disease emerged again and it became clear that the circulating field viruses had changed to circumvent the protection induced by the HVT strain. At this time, a new apathogenic virus was discovered (Rispens strain), which in general has the same serotype as the viruses causing disease. This vaccine strain was introduced very rapidly into the market and produced very good vaccination results.
However, again, after about ten years, new outbreaks of disease occurred; again, circulating field viruses had changed to circumvent the protection induced by the vaccine strain in current use. Then a combination of both vaccines (HVT and Rispens) was used to protect the animals; however, satisfactory results were only seen temporarily. Currently, new outbreaks of disease occur despite all these vaccinations. The reason for this is not yet understood but there is a clear need for the introduction of new potent vaccines.
Problems associated with vaccinations against Marek's disease are that, despite the fact that Marek vaccines have been produced for a long period, the method of preparation of the vaccines could not be improved. The reason for this is that, in general, the essentially host cell-associated virus can be grown essentially only in primary host cells such as in the case of MDV or HVT in primary cells such as fibroblasts prepared from poultry, such as chickens, free of pathogens and, in the case of Varicella Zoster Virus, in (essentially primary) human cells (again, of course, free of contaminating pathogens) and cannot or only with great difficulties be achieved out of context of the specific cell of the respective host. This makes, in general, a vaccine directed against viral infections or disease caused by these types of viruses difficult, if not nearly impossible on a practical level, to produce and thus expensive.
For example, the Rispens vaccine directed against Marek's disease, which is at present considered the only sufficiently potent one, is, as all serotype-1 Marek viruses, strictly host-cell associated. Infectivity of the cell-associated virus (such as, for example, serotype 1 and 2) is completely lost during normal freezing or lyophilization. Therefore, the preparation of the vaccine includes a very complicated and expensive step, where whole cells must be frozen in liquid nitrogen. The vaccine must be stored and transported and kept under liquid nitrogen until use and therefore causes tremendous costs and problems during transport.
Then, at the site of use, the vaccine must be used very carefully, since the infected cells are very sensitive to environmental factors. Factors such as elevated temperatures, exposure to light and residual detergents in glassware used often damage the virus such that no sufficiently viable vaccine batch can be prepared, leading to complete vaccine failures. Such failures can be recognized only when the disease already starts to break out and the affected poultry show symptoms of disease.
In short, up to now, all attempts to provide inactivated, subunit or recombinant vaccines to protect against Marek's disease failed and, therefore, there is currently no alternative to live, cell-associated vaccines comprising Marek's Disease Virus. Marek's disease remains to be controlled by application of infected-cell preparations as a vaccine. These preparations not only contain living cells suspended in DMSO-containing media and the whole variety of cellular antigens, but they also have to be stored in liquid nitrogen. Consequently, the cold chain has to be maintained from vaccine production to the vaccine user and until administration. In addition, once thawed, the vaccine has to be administered within a very short period of time and every bird has to be injected. Several of these problems are shared by those wishing to prepare vaccines against other essentially cell-associated herpes viruses such as Varicella Zoster Virus.
Marek's disease virus (MDV) is a member of the Alphaherpesvirinae subfamily of the Herpesviridae (Lee et al., 2000; Murphy et al., 1995). Based on virulence for the chicken and ability to induce T cell lymphomas, MDV is generally grouped into three serotypes (MDV-1, MDV-2, and MDV-3). MDV-3 represents the herpes virus of turkeys (HVT) which was widely used for vaccination against MDV-related disease. However, after vaccination failures and development of so-called virulent or very virulent MDV-1 (Witter, 1985), attenuated MDV-2 strains and later attenuated MDV-1 strains (e.g., strain CVI 988 Rispens) were used in vaccine formulations (Witter, 1985). In recent years and first reported in the United States, even more virulent MDV-1, so-called very virulent plus (vv+), and MDV-1 variants appeared and caused high incidence of Marek's disease and mortality caused by tumor development and immunosuppression early after infection (Witter, 1997). One vv+ strain, 584A, was passaged more than 100 times on chicken embryo fibroblasts (CEF) and was shown to loose pathogenicity for chickens (Witter, 1997). However, the molecular basis for the increased pathogenicity of vv+ MDV-1 and similarly for loss of virulence are poorly understood because molecular analyses of MDV-1 are difficult to perform. On the one hand, no or only small amounts of infectious virus progeny is released in cultured cells; on the other hand, production of MDV-1 recombinants is laborious and, due to the highly cell-associated nature of the agent in cell culture, multiple rounds of purification of virus recombinants are needed (Cantello et al., 1991; Sakaguchi et al., 1993; Parcells et al., 1994; Schat et al., 1998; Anderson et al., 1998).
On top of that, as mentioned already above, vaccination cannot guarantee to protect the animals from all Marek's disease field viruses. The virus—as all Herpes viruses—is capable of finding ways to escape the immune response induced by the vaccines. Therefore, rapid adaptation of vaccines to the field situation would be needed. Currently, this is done by isolation of field isolates (such as HVT or Rispens) and/or further attenuation in vitro. The isolation itself is causing tremendous problems because of the difficulties of getting the cell-associated infectious virus out of chickens and infecting cells in cell culture. The attenuation steps that would follow are very laborious and time consuming, especially since plaque purification is extremely difficult, which is again due to the cell-associated nature of the virus.
The result from attenuation is normally not defined. As a result of these facts, no vaccines that would provide relief where current HVT- and Rispens-type vaccines fail have entered the market for a long time. In addition, often an over-attenuation occurs during vaccine production since the virus has been passaged for too many times. This further aggravates the low efficacy of the HVT- and Rispens-type vaccines in the field. In short, the following problems constitute a large part of the current impasse in MDV control. There is a low reproducibility of classical vaccine production; one sees over-attenuation of vaccine virus, undefined attenuation of vaccine virus, high production costs, high storage and transport costs, high sensitivity of the vaccine to environmental factors, and a too slow development of new vaccine strains especially for cell associated viruses.
These problems are compounded by the fact that now circulating field viruses give rise to high antibody titers in poultry production stock, whereby these high antibody titers are given through the progeny via maternal antibody in the eggs. The influence of these maternal antibodies during initial infection by current vaccine virus further decreases the current efficacy of vaccination against Marek's disease.